Rescue of high-specificity Cas9 variants using sgRNAs with matched 5’ nucleotides

Abstract

Abstract We report that engineered Cas9 variants with improved specificity—eCas9-1.1 and Cas9-HF1—are often poorly active in human cells, when complexed with single guide RNAs (sgRNAs) with a mismatch at the 5’ terminus, relative to target DNA sequences. Because the nucleotide at the 5’ end of sgRNAs, expressed under the control of the commonly-used U6 promoter, is fixed to a guanine, these attenuated Cas9 variants are not useful at many target sites. By using sgRNAs with matched 5’ nucleotides, produced by linking them to a self-cleaving ribozyme, the editing activity of Cas9 variants can be rescued without sacrificing high specificity.

Keywords

• CRISPR-Cas
• Off-target effect
• Engineered Cas9 variants
• Hammerhead ribozyme-linked sgRNA